Method of treating bladder and lower urinary tract syndromes

ABSTRACT

The present invention relates to bladder and lower urinary tract syndromes, particularly, irrative symptoms, and to a method of treating same using α 1d -adrenergic receptor (α 1d AR) antagonists. The invention further relates to a method of screening compounds for their ability to serve as α 1d AR selective antagonists.

This application is a division of application Ser. No. 10/268,969, filed Oct. 11, 2002, which is a continuation of application Ser. No. 09/306,013, filed May 6, 1999, now abandoned, which claims priority from U.S. Provisional Application No. 60/084,479, filed May 6, 1998, now abandoned, the entire contents of these applications being incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to bladder and lower urinary tract syndromes, particularly, irritative symptoms, and to a method of treating same using α_(1d)-adrenergic receptor (α_(1d)AR) antagonists. The invention further relates to a method of screening compounds for their ability to serve as α_(1d)AR antagonists.

BACKGROUND

Lower urinary tract symptoms (LUTS) resulting from bladder outlet obstruction (BOO) remains one of the most commonly encountered disorders in urology, and can be secondary to fixed anatomical and/or functional causes (Steers et al, Voiding dysfunction: diagnosis, classification, and management, in Adult and Pediatric Urology; Third Edition, J. Y. Gillenwater, et al., Editors. 1996, Mosby-Year Book, Inc.: St. Louis. p. 1220-1325.). Causes of BOO include prostatic enlargement (benign or malignant), bladder neck contracture, urethral stricture, and meatal stricture (Steers et al, Voiding dysfunction: diagnosis, classification, and management, in Adult and Pediatric Urology; Third Edition, J. Y. Gillenwater, et al., Editors. 1996, Mosby-Year Book, Inc.: St. Louis. p. 1220-1325.). Symptoms associated with BOO typically fall into obstructive or irritative categories; obstructive symptoms include hesitancy, poor stream, prolonged urination, and feelings of incomplete emptying, while irritative symptoms consist of frequency, urgency, nocturia, and unstable bladder contractions. The bladder is functionally and anatomically divided into the detrusor (body and ventral base) and trigone (dorsal portion of base extending between the ureteral orifices and the bladder neck) (Zderic et al. Voiding function: relevant anatomy, physiology, pharmacology, and molecular aspects, in Adult and Pediatric Urology; Third Edition, J. Y. Gillenwater, et al., Editors. 1996, Mosby-Year Book, Inc.: St. Louis. p. 1159-1219), with distinct histology, histochemistry, and pharmacology. In contrast, the prostate and trigone have similar vascular supply, innervation, and receptor expression (Gosling et al, Detrusor morphology in relation to bladder outflow obstruction and instability, in Benign Prostatic Hypertrophy, F. Hinman, Editor. 1983, Springer-Verlag: Berlin. p. 666-71).

The physiology of LUTS secondary to benign prostatic hypertrophy (BPH) has two components: (1) a static component related to the increase in prostatic cellular mass and (2) a dynamic component related to variations in prostatic smooth muscle tone (Caine et al, Brit. J. Urol. 47:193-202 (1975)). Histologically BPH is characterized by glandular (epithelial) and stromal (fibromuscular) hyperplasia, with the latter being the dominant factor in the pathogenesis of clinically significant BPH (Shapiro et al, J. Urol. 147: 1293-1297 (1992)). Therefore much attention has focused on the role of the sympathetic nervous system and α₁-adrenergic receptors (α₁ARs) in the dynamic component of BOO, leading to clinical studies of α₁AR antagonists as agents to relieve outlet obstruction. These studies have found that α₁AR antagonists relax prostatic smooth muscle, relieving obstructive symptoms (Chapple, Brit. J. Urol. 1:47-55 (1995), Caine, Urol. Clin. N. Am. 17:641-649 (1990), Kawabe and Niijima, Urol. Int. 42:280-284 (1987), Lepor et al, J. Urol. 148:1467-1474 (1992), Reuther and Aagaard. Urol. Int. 39:312-313 (1984), Matyus and Horvath. Med. Res. Rev. 17:523-535 (1997)). In addition, α₁AR antagonists have been found to relieve the irritative bladder symptoms in men (most often associated with BPH) and women (Matyus and Horvath, Med. Res. Rev. 17:523-535 (1997), Serels and Stein, Neurourol. Urodyn. 17:31-36 (1998)). While it is logical to assume that elimination of BOO would relieve irritative symptoms, a number of recent studies suggest that the relationship between bladder irritability and outlet obstruction is not straightforward (Caine, Urol. Clin. N. Am. 17:641-649 (1990), Chapple and Smith, Brit. J. Urol. 73:117-123 (1994), Steers and De, J. Urol. 140:864-71 (1988), Steers et al, Am. J. Physiol. 266:R20 (1994)).

α₁ARs are members of the larger family of G protein-coupled adrenergic receptors which mediate actions of the endogenous catecholamines norepinephrine (NE) and epinephrine, resulting in smooth muscle contraction. cDNAs encoding three distinct α₁AR subtypes (α_(1a), α_(1b), α_(1d)) have been cloned, expressed in cells, and resultant protein characterized pharmacologically (Schwinn et al, J. Pharmacol. Exper. Ther. 272:134-142 (1995), Hieble et al, Pharmacol. Rev. 47:267-70 (1995)). α_(1a)ARs predominate in prostate and bladder trigone (Price et al, J. Urol. 150:546-551 (1993)), and have been shown to be functionally important in mediating prostate smooth muscle contraction (Forray et al, Mol. Pharmacol. 45:703-708 (1994), Lepor et al., J. Pharmacol. Exper. Ther. 270:722-727 (1994)). In addition to the three cloned α₁AR subtypes which have high affinity for the antagonist prazosin, a fourth type of α₁AR with low affinity for prazosin (α_(1L)) has been postulated (Muramatsu et al, Brit. J. Urol. 74:572-578 (1994)). In spite of initial evidence suggesting a role for the α_(1L)AR in human prostate smooth muscle contraction (Ford et al, Mol. Pharmacol. 49:209-215 (1996)), more recent data suggests RS17053 (the compound used in these studies) detects a low affinity state of the α_(1a)AR in tissues rather than a distinct α_(1L)AR (Ford et al, Br. J. Pharmacol. 121:1127-1135 (1997)). Since non-selective α₁AR antagonists currently used to treat BPH have undesirable side-effects including light headedness, dizziness, and asthenia (Carruthers, Drug Safety 11: 12-20 (1994)), many investigators have suggested that α_(1a)AR subtype selective antagonists might be beneficial in improving BPH-related symptoms via relieving BOO (Matyus and Horvath, Med. Res. Rev. 17:523-535 (1997), Hieble and Ruffolo, Jr., Exp. Opin. Invest. Drugs 6:367-387 (1997)). However, this approach does not take into account that irritative symptoms may persist in spite of relief of outlet obstruction (Hieble and Ruffolo, Jr., Exp. Opin. Invest. Drugs 6:367-387 (1997)).

Very little information exists regarding the role of α₁ARs in human detrusor. One of the few studies addressing this issue suggests human bladder (dome) contains only α_(1a)ARs (Walden et al, J. Urol. 157:1032-1038 (1997)). However, since irritative bladder symptoms persist in some patients despite relief of BOO, nonselective α₁AR antagonists may relieve the irritative effects of BPH through direct effects on bladder detrusor or other sites involved in micturation. The present invention results from the realization that human detrusor expresses two α₁AR subtypes (α_(1d)>α_(1a)). This realization makes possible the identification of α₁AR subtype selective antagonists that can be used to treat irritative symptoms.

SUMMARY OF THE INVENTION

The present invention relates generally to bladder and lower urinary tract syndromes and, more particularly, to a method of identifying α_(1d)AR antagonists that can be used to treat irritative symptoms. The invention also relates to a method of treating irritative symptoms using such agents.

Objects and advantages of the invention will be apparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Schematic of the location of α₁AR subtype probes. Highlighted in bold are regions of α_(1a) (FIG. 1A), α_(1b) (FIG. 1B), and α_(1d) (FIG. 1C) ARs encoded by probes used in RNase protection assays.

FIG. 2. Representative saturation binding isotherm generated using increasing concentrations of the α₁AR radiolabeled antagonist [¹²⁵I]HEAT in human detrusor membranes. Kd is 130±1.09 pM (n=5), similar to that reported for cells stably expressing each cloned human α₁AR subtype (Schwinn et al, J Pharmacol. Exper. Ther. 272:134-142 (1995)—α_(1a/d)AR of the reference refers to the α_(1d)AR subtype described herein since the α₁AR nomenclature used here is the IUPHAR nomenclature (Hieble et al, Phar. Rev. 97:267 (1995)).

FIG. 3. RNase protection assays examining α₁AR subtype expression in detrusor were performed in all patients (n=13). A representative RNase protection assay showing results from five patients is shown. In this experiment, radiolabeled probe for each α₁AR subtype is shown at the far left along with (from left to right) protected fragments resulting from total RNA extracted from rat-1 fibroblast cells stably expressing each cloned human α₁AR subtype (20 mg; positive probe control), yeast tRNA (20 mg; negative control); and total RNA isolated from human detrusor (20 mg) from five patients (lanes 1-5). Gel exposure times are 24 hrs for probe and positive control lanes and 72 hrs for tRNA and human detrusor samples. Although the α_(1d)AR subtype mRNA band is stronger than the α_(1a)AR protected fragment, the α_(1a)AR probe contains 73% more radiolabeled αUTP compared with the α_(1d); hence, after normalization for radioactive label incorporation, two-fold predominance of the α_(1d)AR subtype in human detrusor is apparent.

FIGS. 4A and 4B. Results from RT-PCR experiments on human detrusor (FIG. 4A) and rat whole bladder (FIG. 4B) RNA. α₁AR subtype specific cDNA in plasmid vectors served as positive controls.

FIG. 5. α₁AR subtype expression in human detrusor was determined using competition analysis with the α_(1d)AR-subtype selective ligand BMY7378. Results from a representative curve are shown demonstrating a two-site fit with high affinity Ki corresponding to the α_(1d)AR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the recognition of α_(1d)AR as the α₁AR subtype responsible for irritative symptoms associated with bladder and lower urinary tract diseases. The invention provides, in one embodiment, a method of selecting α_(1d)AR antagonists and, in a further embodiment, a method of treating irritative symptoms using α_(1d)AR antagonists. (The nomenclature used herein is the new nomenclature provided in Hieble et al, Phar. Rev. 97:267 (1995)).

The method of treatment to which the invention relates comprises administering to a patient suffering irritative symptoms an amount of an α_(1d)AR antagonist sufficient to relieve such symptoms. In accordance with the invention, irritative symptoms include excessive frequency of urination, urgency of urination, nocturia and unstable bladder contractions. Patients amenable to treatment include men and women, children and adults. In males, a preferred antagonist is both an α_(1a)AR and an α_(1d)AR antagonist. In females, preferred antagonists are α_(1d)AR specific antagonists. The amount of the antagonist to be administered and the treatment regimen will vary with the antagonist, the patient and the effect sought. Optimum doses and regimens, however, can be readily determined by one skilled in the relevant art.

The present invention also relates to a method of screening compounds for their ability to bind primarily to α_(1d)AR and thereby to function, potentially, as α_(1d)AR antagonists Preferred α_(1d)AR selective antagonists show at least a two fold selectivity for α_(1d)AR relative to α_(1a)AR or α_(1b)AR. Binding assays of this embodiment invention include cell-free assays in which α_(1d)AR, or portion thereof (e.g. relevant transmembrane portion—see, generally, Hwa et al, J. Biol. Chem. 271:7956 (1996)). is incubated with a test compound (proteinaceous or non-proteinaceous) which, advantageously, bears a detectable label (e.g., a radioactive or fluorescent label). Preparations of membranes that bear α_(1d)AR can be used in this assay, including commercially available preparations (e.g. the NEN multireceptor kit (NET 1034)). Following incubation, the α_(1d)AR, or portion thereof, free or bound to test compound, can be separated from unbound test compound using any of a variety of techniques (for example, the α_(1d)AR (or portion thereof) (e.g., associated with a membrane) can be bound to a solid support (e.g., a plate or a column) and washed free of unbound test compound). The amount of test compound bound to α_(1d)AR, or portion thereof, is then determined using a technique appropriate for detecting the label used (e.g., liquid scintillation counting in the case of a radiolabelled test compound). (See Schwinn et al, J. Pharm. Exp. Ther. 272:134 (1995).)

Binding assays of this embodiment can also take the form of cell-free competition binding assays. Such assays can be conducted as described in the Examples that follow (see particularly Example 2 (the test compound being substituted for BMY 7378)—see also Schwinn et al, J. Pharm. Exp. Ther. 272:134 (1995)). Alternatively, α_(1d)AR, or portion thereof, can be incubated with a compound known to interact, specifically, with α_(1d)AR (e.g., BMY7378), which compound, advantageously, bears a detectable label (e.g., a radioactive or fluorescent label). A test compound (proteinaceous or non-proteinaceous) is added to the reaction and assayed for its ability to compete with the known (labeled) compound for binding to α_(1d)AR, or portion thereof. Free known (labeled) compound can be separated from bound known compound, and the amount of bound known compound determined to assess the ability of the test compound to compete. This assay can be formatted so as to facilitate screening of large numbers of test compounds by linking the α_(1d)AR, or portion thereof or, to a solid support so that it can be readily washed free of unbound reactants.

α_(1d)AR, or portion thereof, suitable for use in the cell-free assays described above can be isolated from natural sources (e.g., as membrane preparations derived from bladder, e.g., human bladder) or prepared recombinantly or chemically. The α_(1d)AR, or portion thereof, or can be prepared as a fusion protein using, for example, known recombinant techniques. Preferred fusion proteins include a HIS tag, a FLAG tag, a GFP tag or other tag (moiety) suitable for use in colorimetric assays. Typically, the non-α_(1d)AR moiety is present in the fusion protein N-terminal to the α_(1d)AR, or portion thereof domain, but it can also be C-terminal.

As indicated above, the α_(1d)AR, or portion thereof, can be present linked to a solid support, including a plastic or glass plate or bead, a chromatographic resin, a filter or a membrane. Methods of attachment of proteins, or membranes containing same, to such supports are well known in the art.

The binding assays of the invention also include cell-based assays in which α_(1d)AR, or portion thereof, is associated with the cell membrane of an intact cell. Cells suitable for use in such assays include cells that naturally express α_(1d)AR and cells that have been engineered to express, advantageously, over express, α_(1d)AR (or portion thereof). Advantageously, cells expressing human α_(1d)AR are used. Suitable cells are preferably eucaryotic, including mammalian (human and nonhuman) cells, insect cells and yeast cells.

Cells can be engineered to express α_(1d)AR (advantageously, human α_(1d)AR, or portion thereof) by introducing into a selected host (e.g. a eucaryotic host) an expression construct comprising a sequence encoding α_(1d)AR, or portion thereof, operably linked to a promoter. A variety of vectors and promoters can be used. (See Schwinn et al, J. Pharm. Exp. Ther. 272: 134 (1995).) Introduction of the construct into the host can be effected using any of a variety of standard transfection/transformation protocols (see Molecular Biology, A Laboratory Manual, second edition, J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbor Press, 1989). Cells thus produced can be cultured using established culture techniques suitable for the involved host. Culture conditions can be optimized to ensure expression of the α_(1d)AR (or portion thereof) encoding sequence.

While for the cell-based binding assays it is appropriate that the α_(1d)AR (or portion thereof) be associated with the cell membrane, for other purposes the expression product can be secreted into the culture medium or present in the cell cytoplasm.

The cell-based binding assays of the invention can be carried out essentially as described above with respect to the cell free assays. Advantageously, the cell used expresses predominantly the α_(1d)AR subtype. By way of example, the cell-based binding assay can be carried out by adding test compound (advantageously, bearing a detectable (e.g., radioactive or fluorescent) label), to medium in which the α_(1d)AR (or portion thereof) expressing cells are cultured, incubating the test compound with the cells under conditions favorable to binding and then removing unbound test compound and determining the amount of test compound associated with the cells.

As in the case of the cell-free assays, the cell-based assays can also take the form of competitive assays, as described above. For example, a compound known to bind α_(1d)AR (and preferably labelled with a detectable label) can be incubated with α_(1d)AR (or portion thereof expressing cells in the presence and absence of test compound. The affinity of a test compound for α_(1d)AR can be assessed by determining the amount of known compound associated with the cells incubated in the presence of the test compound, as compared to the amount associated with the cells in the absence of the test compound.

A test compound identified in one or more of the above-described assays as being capable of binding to α_(1d)AR can, potentially, serve as an α_(1d)AR antagonist and therefore be suitable for use in the irritative symptom treatment method of the invention. To determine the specific effect of any particular test compound selected on the basis of its ability to bind α_(1d)AR, various assays can be used including IP assays (see Schwinn et al, J. Pharm. Exp. Ther. 272:134 (1995)) and bladder (e.g. human bladder) smooth muscle contraction assays (Ford et al, Mol. Pharm. 49:209 (1996)). Compounds suitable for use in treating irritative symptoms will be associated with antagonistic (inhibitory) effects in the IP assay and contraction inhibitory effects in the contraction assay.

In another embodiment, the invention relates to compounds identified using the above-described assays as being α_(1d)AR antagonist. The compounds identified in accordance with the above assays can be formulated as pharmaceutical compositions. Such compositions comprise the compound and a pharmaceutically acceptable diluent or carrier. The compound can be present in dosage unit form (e.g., as a tablet or capsule) or as a solution, preferably sterile, particularly when administration by injection is anticipated. The dose and dosage regimen will vary, for example, with the patient, the compound and the effect sought. Optimum doses and regimens can be determined readily by one skilled in the art.

In another embodiment, the invention relates to antibodies specific for α_(1d)AR, and antigen binding fragments thereof, including F(ab)₂′ or F(ab) fragments. The antibodies can be monoclonal or polyclonal and can be prepared using standard techniques. The antibodies can be used in α_(1d)AR purification protocols or the antibodies can be formulated as pharmaceutical compositions and used therapeutically as α_(1d)AR antagonists.

In yet another embodiment, the present invention relates to a gene therapy approach to treating irritative symptoms. In this embodiment, oligonucleotides (constructs) are used that, upon administration, result in the production of a molecule that down regulates production of α_(1d)AR. In a related embodiment, the present invention relates to α_(1d)AR antisense constructs and to a method of using same to treat irritative symptoms. Such constructs can be designed to target any of a variety of regions of the α_(1d)AR gene, including the encoding sequence (e.g., regions encoding the intracellular portion that interacts with G protein and participates in the signal transduction pathway) and the 5′-untranslated region.

Delivery of the above-described constructs can be effected using any of a variety of approaches, including installation into the bladder (e.g. via the uretha) and introduction into the cerebrospinal fluid. The constructs can also be administered systemically, in which case targeting can be effected using, for example, smooth muscle (e.g. bladder smooth muscle) specific promoters.

Effective vectors for use in the above-described gene therapy/antisense embodiments include viral vectors, such as retroviral vectors, adenoviral vectors and adenoassociated viral vectors. The constructs can also be present in association with a lipid, e.g. a liposome. (For details of antisense constructs and delivery systems, etc. see, for example, Wagner Nature 372:333 (1994).) The amount of construct to be administered will vary, for example, with the construct, the patient and the effect sought. One skilled in the relevant art can readily optimize the dose and treatment regimen.

In yet another embodiment, the invention relates to kits, for example, kits suitable for conducting assays described herein. Such kits can include α_(1d)AR, or portion thereof, for example, bound to a solid support. The kit can include an α_(1d)AR-encoding sequence, α_(1d)AR antisense construct or α_(1d)AR-specific antibody. The kit can include any of the above components disposed within one or more container means. The kit can further include ancillary reagents (e.g., buffers) for use in the assays.

Certain aspects of the present invention are described in greater detail in the non-limiting Examples that follow.

EXAMPLES

The following experimental details are relevant to the specific Examples that follow.

Tissue preparation. Full-thickness human bladder detrusor was obtained as discarded “normal” tissue adjacent to tumor specimens (n=1 radical cystectomy, n=12 radical cystoprostatectomy for transitional cell carcinoma of the bladder) with appropriate institutional approval. Each sample was inspected by a pathologist, and normal tissue confirmed. Detrusor smooth muscle was grossly teased from urothelial and serosal layers, snap frozen in liquid nitrogen within 30 minutes of excision, and stored at −70° C. for later use. Whole rat bladder was obtained from euthanized male Sprague-Dawley rats (Charles River Laboratories; Wilmington, Mass.) with institutional animal care committee approval. Rat tissue was harvested within two minutes of death, snap frozen in liquid nitrogen, and stored at −70° C. for later use.

Human detrusor and rat bladder membrane preparation. Human detrusor and rat whole bladder was minced over dry ice, and suspended in cold lysis buffer (5 mM Tris HCl and 5 mM EDTA, pH 7.4) with protease inhibitors benzamidine (10 mg/ml), leupeptin (5 mg/ml), and soybean trypsin inhibitor (10 mg/ml) (Sigma Chemical Company; St. Louis, Mo.). A lysate was prepared with a Polytron PT 3000 (Brinkmann; Westbury, N.Y.) at 10,000 rpm for 10 seconds. After pelleting at 40,000×g for 15 minutes (Sorvall SM24 rotor), membranes were suspended in cold resuspension buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, pH 7.4) with protease inhibitors, and kept on ice for immediate use (or stored at −70° C. for later use). Protein content was determined using the bicinchoninic assay (BCA) with bovine serum albumin (BSA) standards (Pierce; Rockford, Ill.).

Radioligand binding. All mRNA and protein studies described were performed using detrusor from each patient described above (n=13). In order to conserve sample, and yet fully characterize α₁ARs in human detrusor, additional full saturation binding isotherms were generated in human detrusor samples from a subset of patients (n=5) using a buffer consisting of 150 mM NaCl, 50 mM Tris-HCl and 5 mM EDTA, pH 7.4, with protease inhibitors. Each reaction was performed in triplicate, in a total volume of 0.25 ml, including diluted human detrusor membranes (50 to 100 mg protein) and the α₁AR antagonist [¹²⁵I]HEAT (NEN Research Products-DuPont; Boston, Mass.) ranging in concentration from 2-900 pM; nonspecific binding was measured in the presence of 1 mM prazosin (Sigma). The reaction proceeded at 25° C. for 45 minutes, and was terminated with five-fold dilution of ice-cold 50 mM Tris HCl, pH 7.4 buffer, followed by rapid filtration over GF/C filters using a Brandel harvester. Dried filters were then counted in a gamma counter. Specific binding was calculated by subtracting nonspecific binding from total binding. Saturation curves were fit with noniterative regression analysis using InPlot software (GraphPad; San Diego, Calif.). Total α₁AR density was then determined in each detrusor sample as described above, using a saturating concentration of [¹²⁵I]HEAT (300 pM). Results are reported as mean±SEM to two significant figures.

To determine Ki values in human detrusor for α₁AR subtype discriminating ligands, competition binding was performed in triplicate in a total volume of 0.25 ml using binding buffer (see saturation binding above). Human detrusor membranes (50 to 100 μg protein) were incubated with a K_(d) concentration (120 pM) of the α₁AR antagonist [¹²⁵I]HEAT, and increasing concentrations (10⁻¹² to 10⁻³M) of the non-radiolabeled α_(1d)AR-selective ligand BMY7378 (Research Biochemicals International; Natick, Mass.). Reaction conditions were as described above. Curves were fit with noniterative regression analysis using InPlot software (GraphPad).

Preparation of RNA. Total RNA was extracted from human detrusor or rat whole bladder samples using the RNazol method (Tel-Test, Inc.; Friendswood, Tex.). RNA was quantitated using a spectrophotometer at 260/780 nm, and aliquoted into 20 mg samples for immediate use.

Human α₁AR cDNA constructs. The human α_(1a)AR probe consists of a 0.326 kb (PvuII/HindIII) fragment in pGEM-4Z (Promega Corporation; Madison, Wis.), corresponding to nucleotides 958-1283 of the cloned human α_(1a)AR cDNA (GenBank #L31774). The human α_(1b)AR probe consists of a 0.673 kb (XhoI/BamHI) fragment in pGEM-4Z (Promega), corresponding to nucleotides 94-766 of the cloned human α_(1b)AR cDNA (GenBank #L31773). The human α_(1d)AR probe consists of a 0.377 kb (EcoRI/PstI) fragment, corresponding to nucleotides 520-896 of the cloned human α_(1d)AR cDNA (GenBank #L31772). FIG. 1 shows the location of each α₁AR subtype probe within a schematic of the encoded protein. The human cyclophilin probe consists of a 0.103 kb (KpnI/EcoRI) fragment in pTRI (Ambion, Inc.; Austin, Tex.), corresponding to nucleotides 38-140 of the cloned human cyclophilin gene (GenBank #X52856).

Labeling of RNA probes. Antisense single-stranded radiolabeled RNA probes were generated from linearized α₁AR cDNA constructs using RNA polymerase T7 (α_(1a), cyclophilin) and SP6 (α_(1b), α_(1d)) as described in the Promega Protocols and Applications Guide (Promega Corporation; Madison, Wis.). α_(1a)AR and α_(1d)AR cDNA constructs were linearized with EcoRI, and the α_(1b)AR cDNA construct was linearized with HindIII. ³²P-αUTP (NEN Research Products-DuPont) was incorporated into RNA probes at the time of probe synthesis. All probes were purified on a 5% polyacrylaminde gel (300V for 1.5 hr); after exposure to film for 3 min, radiolabeled RNA probes were excised from the gel and passively eluted overnight into 400 μl of RPA II kit (Ambion) elution buffer at 37° C.

RNase protection assays. RNase protection assays were conducted as previously described (Zinn et al, Cell 34:865-879 (1983)) with a few modifications. In brief, total RNA samples (20 mg) were dissolved in 20 ml of hybridization buffer containing >20-fold excess of radiolabeled probe (2×10⁵ cpm/reaction for α_(1a), α_(1b), α_(1d), and 1×10⁵ cpm/reaction for cyclophilin), and incubated overnight at 55° C. (α_(1a), α_(1b)) and 65° C. (α_(1d), cyclophilin). To ensure specificity of the synthesized radiolabeled antisense human α₁AR subtype selective probes, RNase protection assays were performed in tandom with total RNA extracted from rat-1 fibroblast cells stably expressing each cloned human α₁AR subtype. As a negative control, RNase protection assays for each α₁AR subtype selective probe were performed in tandom with yeast tRNA samples and other non-hybridizing α₁AR subtypes. Antisense radiolabeled probe to the highly conserved region of the constitutively expressed human cyclophilin gene was also utilized as a control to ensure identical amounts of total RNA in each assay. The final gel was exposed to X-Omat AR film (Eastman Kodak Company; Rochester, N.Y.) for 24-72 hours.

α₁AR mRNA quantitation in human detrusor smooth muscle from RNase protection assays. In order to quantitate relative α₁AR subtype mRNA, each RNase protection assay final gel was exposed to PhosphorImager plates (Molecular Dynamics; Sunnyvale, Calif.) for 24 hours. Volume integration of specific protected radiolabeled bands for each mRNA resulting from hybridization products was corrected for background, normalized for cyclophilin signal, and expressed as arbitrary density units, using ImageQuant gel image-analysis software (Molecular Dynamics). α₁AR probes contained the following number of UTP sites for ³²P-αUTP incorporation: α_(1a) 88, α_(1b) 117, α_(1d) 51. Arbitrary density units were normalized to the lowest ³²P-αUTP incorporating probe (α_(1d)AR) and then expressed as a fraction (±SEM) of total α₁AR mRNA signal strength.

Polymerase Chain Reaction (PCR). RT-PCR was used to confirm expression of human detrusor α₁AR subtypes (to ensure low concentrations of a subtype were not missed in human bladder) and to compare α₁AR subtype mRNA expression in rat whole bladder with previously published rat data (Walden et al, J. Urol. 157:1032-1038 (1997), Scofield et al, J. Pharmacol. Exper. Ther. 275:1035-1042(1995)). Human and rat α₁AR subtype primers were synthesized at Duke University Medical Center. Reverse transcription of 1 mg of DNase-treated human detrusor or rat bladder RNA was performed in triplicate in a 20 ml reaction mixture containing 5 mM MgCl₂, 1 mM each of dATP, dCTP, dGTP, and dTTP, 10 mM TrisHCl, 50 mM KCl, 2 ml DEPC treated water, 2.5 mM random hexamers, 1 unit of RNase inhibitor, and 2.5 units of MuLV Reverse Transcriptase (Perkin Elmer; Foster City, Calif.); simultaneous control samples not treated with reverse transcriptase were used to rule out amplification of genomic DNA. Reverse transcriptase reactions were run for 60 min at 42° C. 5 min at 95° C., and 10 min at 4° C. Each α₁AR mRNA subtype was amplified by PCR in triplicate in a 100 ml reaction containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2 mM MgCl₂, 200 mM each of dATP, dCTP, dGTP and dTTP, 15 pM of sense and antisense primer. 5% DMSO, and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer). PCR reactions were performed in a DeltaCycler II™ temperature cycler (ERICOMP; San Diego, Calif.). The following conditions were established for all three rat primer sets: one denaturation cycle for 3 minutes at 95° C., 35 cycles of 1 min at 95° C., 1 min annealing at 58° C. and a 1 min extension at 72° C. The following conditions were established for all three human primer sets: one denaturation cycle for 3 minutes at 95° C., 35 cycles of 1 min at 95° C. 1 min annealing at 60° C. for α_(1a) and α_(1b), and 68° C. for α_(1d), and a 1 min extension at 72° C. A final extension cycle was performed for 10 min at 72° C. Reaction mixtures were then cooled at 4° C. 10 ml of each PCR product was separated by gel electrophoresis in 0.8% agarose. Since PCR experiments were only confirmatory in nature by design, exact quantitation (requiring competitive PCR) was not performed. However, to ensure that any statement regarding relative mRNA levels is appropriate, it is important to note that conditions described above (e.g. different annealing temperatures) were chosen after extensive preliminary analysis with each primer set to ensure optimal amplification conditions with similar primer product efficiency. Equality of reverse transcription efficiency for products was checked using equal concentrations of starting control cDNA; these reactions also served as a positive control for use of correct primer sets. Thirty-five cycles of amplification was chosen since it is at the upper end of the linear amplification range for all six primer sets (α₁AR mRNAs are rare at baseline in many human tissues and in our hands 40 cycles of amplification is where the curve becomes non-linear).

Example 1 Human Patient Population

Human detrusor smooth muscle was obtained from male (n=12) and female (n=1) patients. Patient age ranged from 56 to 76 years old (mean=59.6). Significant past medical history included tobacco abuse, coronary artery disease, hypertension controlled with α₁AR or βAR antagonists (n=3), and a history of BOO (n=2) necessitating previous transurethral resection of prostate. Comparison of results from patients with hypertension and/or BOO (n=5) suggests medical history did not affect the results. A larger study would be required to make any definitive statement in this regard.

Example 2 α₁AR Ligand Saturation Binding

Pharmacological characteristics of α₁ARs in human detrusor include a Kd for the radiolabeled α₁AR antagonist [¹²⁵I]HEAT of 130±1.9 pM, similar to that reported for cells stably expressing the cloned human α_(1a)AR subtype ((Schwinn et al, J Pharmacol. Exper. Ther. 272:134-142 (1995)). A representative saturation binding isotherm is shown in FIG. 2. Total α₁AR density as measured by saturation binding in human detrusor membrane preparations with the α₁AR antagonist [¹²⁵I]HEAT is 6.3±1.0 fmol/mg protein (mean±SEM, range 2.7-9.0, n=13). Although low (with corresponding high non-specific binding of 70-80% as expected), α₁AR expression is reproducible and consistent within and between patients.

Example 3 Identification and Quantification of the α₁AR mRNA Subtypes in Human Bladder Detrusor

In order to determine which α₁AR subtypes are present in human detrusor, molecular approaches were chosen due to their sensitivity and specificity. To ensure specificity of the synthesized radiolabeled antisense human α₁AR subtype selective probes, RNase protection assays were performed simultaneously with total RNA extracted from rat-1 fibroblast cells stably expressing each cloned human α₁AR subtype. Each α₁AR subtype specific probe protects a single predominant fragment of predicted size without cross-hybridization (FIG. 3, positive control cells); a lack of cross-hybridization between subtypes with each probe (Price et al, Mol. Pharmacol. 46:221-226 (1994)). As was previously demonstrated a further negative control, RNase protection assays for each α₁AR subtype selective probe were performed in tandom with yeast tRNA samples, where no hybridization is demonstrated (FIG. 3, tRNA lane). Human detrusor contains α_(1d)AR>α_(1a)AR mRNA, but no α_(1b)AR mRNA in every patient studied (n=13; FIG. 3 shows representative results from patients number 1 through 5). This data, when corrected for background, normalized for cyclophilin content, and corrected for probe ³²P-αUTP incorporation, reveals that α_(1d)AR mRNA constitutes 66±4.8% and α_(1a)AR mRNA 34±4.8% of the total α₁AR mRNA in human detrusor.

Example 4 Confirmation of α₁AR Subtype mRNA in Human Detrusor and Comparison with Rat Whole Bladder Using RT-PCR

In order to confirm results from RNase protection assays and to compare with another frequently used animal model (rat), α₁AR subtype expression was examined using RT-PCR in each patient. Primer nucleotide sequences, melting temperatures (T_(m)), and primer positions relative to the cDNA sequence are shown in Table 1 and Table 2; these primers do not span an intron.

TABLE 1 Oligonucleotide primers used for rat α₁AR subtype RT-PCR. primer position Rat α₁AR nucleotide sequences relative primers 5′ 3′ T_(m) to cDNA α_(1a)AR sense GTAGCCAAGAGAAAGCCG 62° C. 628-647 α_(1a)AR CAACCCACCACGATGCCCAG 66° C. 839-820 antisense α_(1b)AR sense GCTCCTTCTACATCCCGCTCG 68° C. 629-649 α_(1b)AR AGGGGAGCCAACATAAGATGA 62° C. 928-908 antisense α_(1d)AR sense CGTGTGCTCCTTCTACCTACC 66° C. 759-779 α_(1d)AR GCACAGGACGAAGACACCCAC 68° C. 1062-1042 antisense The nucleotide sequences listed above correspond to the following sequence identifiers, respectively: SEQ ID NOs: 1-6.

TABLE 2 Oligonucleotide primers used for human α₁AR subtype RT-PCR. primer position Human α₁AR nucleotide sequences relative primers 5′ 3′ T_(m) to cDNA α_(1a)AR sense ATCATCTCCATCGACCGCTACA 66° C. 355-376 α_(1a)AR TCACTTGCTCCGAGTCCGACTT 68° C. 697-676 antisense α_(1b)AR sense GCTCCTTCTACATCCCTCTGG 68° C. 629-649 α_(1b)AR AGGGTAGCCAGCACAAGATGA 67° C. 928-908 antisense α_(1d)AR sense ACCACGCGCAGCCTCGAGGCAGGC 84° C. 850-873 α_(1d)AR GAGCGAGCTGCGGAAGGTGTGGCC 82° C. 999-976 antisense The nucleotide sequences listed above correspond to the following sequence identifiers, respectively: SEQ ID NOs: 7-12.

Although RNase protection assays are considered the “gold standard” for quantitating mRNA present in a given tissue, this approach is not as sensitive as PCR, therefore very small amounts of mRNA can be missed in a RNase protection assay but demonstrated by PCR. As shown in FIG. 4, RT-PCR performed on human detrusor total RNA demonstrates the presence of α_(1a)AR and α_(1d)AR mRNA, and lack of α_(1b)AR mRNA, consistent with data from the RNase protection assays. Of note, α_(1d)AR mRNA accounts for approximately 60-70% of total α₁AR mRNA in human detrusor with α_(1a)AR mRNA accounting for 30-40%, again confirming the RNase protection assay results. Species heterogeneity (human versus rat) of α₁AR subtype mRNA expression has been previously reported for many tissues (Price et al, Mol. Pharmacol. 46:221-226 (1994), Price et al, Mol. Pharmacol. 45:171-175 (1994)). Indeed, as seen in FIG. 4, RT-PCR performed on pooled rat bladder total RNA demonstrates the presence of all three α₁AR mRNAs in roughly equal concentrations in rat.

Example 5 Determination of α₁AR Subtype Expression at a Protein Level

Competition analysis was used to determine α₁AR subtype expression at a protein level in human detrusor. Since molecular studies demonstrate a predominance of α_(1d)AR mRNA, the α_(1d)AR-selective compound BMY7378 was used in these studies. As graphically represented in FIG. 5, a two-site fit was evident in every patient studied (n=13), with high affinity binding predominating (high pK_(i)=8.6±0.2 [66±3.1% total] vs. low pK_(i)=4.9±0.2 [35±3.1% total]) (Table 3).

TABLE 3 Results from competition binding experiments utilizing membranes from rat-1 fibroblasts stably transfected with each α₁AR subtype (controls) and human detrusor (n = 13). Since no α_(1b)AR was found in human detrusor by RNase protection assays and RT-PCR, one versus two site fit of the data was utilized. BMY7378 (pK_(i)) α_(1a)AR α_(1b)AR α_(1d)AR % High % Low Human detrusor 4.9 ± 0.2 — 8.6 ± 0.2 66 ± 3.1 35 ± 3.1 Control α_(1a) 4.8 ± 0.1 — — Control α_(1b) — 5.1 ± 0.3 — Control α_(1d) — — 8.5 ± 0.1

All documents cited above are hereby incorporated in their entirety by reference.

One skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. 

1. A method of screening for a candidate agent suitable for use in relieving irritative symptoms of bladder or lower urinary tract disease in a patient comprising: i) incubating a test compound with α_(1d)-adrenergic receptor (α_(1d)AR), or portion thereof, ii) determining the amount of said test compound bound to said α_(1d)AR, or portion thereof, and iii) selecting a test compound that binds said α_(1d)AR, or portion thereof, as said candidate agent for use in relieving irritative symptoms of bladder or lower urinary tract disease in a patient.
 2. The method according to claim 1 wherein said test compound bears a detectable label.
 3. The method according to claim 1 wherein said α_(1d)AR, or portion thereof, is present in a cell membrane.
 4. The method according to claim 3 wherein said membrane is the membrane of an intact cell.
 5. The method according to claim 4 wherein said cell is a eucaryotic cell.
 6. The method according to claim 4 wherein said cell is a cell that has been engineered to express or over-express said α_(1d)AR, or portion thereof.
 7. The method according to claim 1 wherein said test compound is incubated with said α_(1d)AR, or portion thereof, in the presence of a known α_(1d)AR binding agent (α_(1d)BA) and the amount of said test compound that binds to said α_(1d)AR, or portion thereof, is determined indirectly by determining the amount of said known α_(1d)BA that binds to said α_(1d)AR, or portion thereof.
 8. The method according to claim 7 wherein said known α_(1d)BA bears a detectable label.
 9. The method according to claim 7 wherein said known α_(1d)BA is BMY7378 or tamsulosin.
 10. The method according to claim 1 wherein said portion comprises the transmembrane portion of α_(1d)AR.
 11. A method of screening for a candidate agent suitable for use in relieving irritative symptoms of bladder or lower urinary tract disease in a patient comprising: i) incubating a test compound with α_(1d)-adrenergic receptor (α_(1d)AR), or portion thereof comprising the transmembrane domain of α_(1d)AR, ii) determining the amount of said test compound bound to said α_(1d)AR, or said portion thereof, iii) identifying a test compound that binds said α_(1d)AR, or said portion thereof, and iv) selecting a test compound that binds said α_(1d)AR, or portion thereof, as said candidate agent for use in relieving irritative symptoms of bladder or lower urinary tract disease in a patient. 